The aim of our project is to use analytical techniques to identify the existence of Lyme borreliosis, commonly known as Lyme Disease in Ticks and investigate the distribution of Lyme borreliosis in the Dorset area. Results are presented from successful and accurate gel electrophoresis using Safe Blue indicator; although only one positive result for the disease was identified, the skills we learnt while completing this project were invaluable.
Lyme borreliosis, is caused by spirochetal bacteria from the genus Borrelia. The official species is Borrelia burgdorferi. Spirochetes are surrounded by peptidoglycan and flagella, along with an outer membrane. The spiral-shaped, bacteria have flexible, pliable bodies that are moved by organs called flagella. These are long lash-like appendages that protrudes from the cells’ body. With the aid of flagella protein they rotate allowing the bacteria to move in its host environment. The bacterium is different from any other bacteria known due to its unique structure; it has a three-layer cell wall which helps determine the spiral shape of the bacteria. It also has a clear gel-like coat of glyco-proteins which surround the bacteria which acts as protection to prevent the bacteria from being detected by the immune system. Therefore, the disease is hard to identify in humans. Furthermore, the bacteria have replications of specific genes known as “Blebs” which are released from the bacterium into the host and can irritate the immune system. Finally, when entering a cell, the bacteria releases digestive enzymes that dissolve the cell. When targeted at the immune system and T-lymphocytes the immune response is weakened and the disease is even harder to identify. When bitten by an infected tick the microbes travel through the bloodstream to the heart. Ticks are infected by deer and other mammals causing large numbers to accumulate in singular areas thus making the threat to humans extremely dangerous, but just how dangerous is it to those living in the Dorset area? ((Grier, T,M., 1997. “The Complexeities of Lyme Disease.” [online] Available from: https://www.lymeneteurope.org/info/the-complexities-of-lyme-disease Accessed August 9th, 2017.))
Gathering the Ticks
The ticks were sent in by volunteers across the Dorset area, we ended up with over 400 ticks, these were randomly selected to avoid bias into a sample of 120. The ticks were then profiled and recorded for future reference. This included their sex, size and developmental stage of the ticks. Female ticks are generally larger and have dark brown head and upper body while males are smaller ((Island, Unknown The University of Rhode. n.d. Tick encounter resource page. [online] Available from: http://www.tickencounter.org/tick_identification/dog_tick Accessed August 9th, 2017.)) The ticks were then assigned given numbers to identify them.
(Above: Photograph of me and a fellow student identifying PTC and our results. The Lane with 2 contains belongings to a person with Sickle Cell Anaemia)
We began our project by identifying the PTC allele and Sickle Cell Anaemia in anonymous volunteers to develop and perfect our skills. This was crucial as the DNA is easily contaminated and the method requires extreme precision. Without this shorter investigation, the results we obtained would not have been as reliable as they were.
(Above: The gel electrophoresis for Sickle Cell Anaemia. It is the second lane from the left. Sickle Cell Anaemia only has two bars as it recessive, and can be identified easily in this way)
How does PCR work?
Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence”. ((University of Utah. n.d. Learn.Genetics. [online] Available from:http://learn.genetics.utah.edu/content/labs/pcr/. Accessed August 9th, 2017.)) Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycling commonly consisting of two or three discrete temperature steps. ((Rychlik W, Spencer WJ, Rhoads RE. 1990. Optimization of the annealing temperature for DNA application in vitro.)) DNA primers are short sections of DNA that are complementary to the three prime ends of the segment of DNA you want to elongate. Through complementary base pairing one primer attaches to the top strand and the other to the bottom. DNA polymerase, a naturally occurring complex of proteins, attaches to the primer and starts to add nucleotides to it, thus creating copies of the DNA segment. ((University of Utah. n.d. Learn.Genetics. [online] Available from:http://learn.genetics.utah.edu/content/labs/pcr/. Accessed August 9th, 2017.)) Tick DNA primer is the cytochrome 6 oxidase primer and the Borrelia DNA primer is the B.burgdorferi 16s rRNA primer and the 129 base product. By the 35th cycle there is 68 billion copies of the DNA segment that can be analysed by Gel electrophoresis.
How does Gel Electrophoresis work?
Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA according to their size. Charged molecules move through a gel when an electric current is passed across it. An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge. The movement of charged molecules is called migration, molecules migrate towards the opposite charge; a molecule with a negative charge will therefore be pulled towards the positive end. The gel consists of a permeable matrix through which molecules can travel when an electric current is passed across it. Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance. As a result, the molecules are separated by size. ((Unknown. 2016. Your Genome. [online] Available from:https://www.yourgenome.org/facts/what-is-gel-electrophoresis Accessed August 9th, 2017.)) DNA is made up of molecules called nucleotides. Each nucleotide contains a phosphate group, a sugar group and a nitrogen base; adenine (A), thymine (T), guanine (G) and cytosine (C). ((Rettner, RR., 2013. DNA: Definition, Structure and Discovery [online] Available from: https://www.livescience.com/37247-dna.html Accessed August 9th, 2017.)) A phosphate group is a functional group or radical comprised of phosphorus attached to four oxygen atoms, and with a net negative charge, thus represented as PO4- (Biology Online). It is due to the negatively charged phosphate group that the DNA molecule as a whole is polar and travels from the negative end of the chamber to the positive. The DNA ladder contains bands at known lengths, using a positive Borrelia control you can see what length the Borrelia gene travels to against the ladder and thus if we have any positive results from our Ticks as they will be found at the same length.
After 2 years’ worth of analysing ticks from across the Dorset area we only found one positive result for Lyme disease. The lanes 1 and 8 are DNA ladders, and lane 2 is a positive result for Lyme disease. In Lane 5 there is one band for tick DNA and just above there is faint band at the same distance as the positive result, meaning this tick had Lyme disease. The Borrelia gene is lighter and thus travelled further than the tick DNA and the result is also fainter. The tick was number 48 and was donated to us from the location marked on the map.
(Above: The distribution of Ticks we collected)
From our results, we had 1 out of 120 test positively for the results, our results were reliable due to extreme caution taken during the experiment and trustworthy equipment such as pipettes allowing us to accurately measure in micrometers. Our results show that Lyme disease is not at all common in the Dorset area and that the stigma surrounding it of being “deadly and frequent” is inaccurate as the chances of finding an infected tick is 1/120 from our research. This means that tourist and nature enthusiasts are free to walk the scenic landscape of Dorset without worrying about the threat of Lyme Disease.
(Above: the gel electrophoresis of our only positive result, the double band is in Lane 5 and aligns with the positive control in Lane 2.)
About the Author
Madeleine is a 17 year old A-Level student at the Thomas Hardye School. She is working towards studying molecular biology at university while her current subjects are: Biology, Chemistry, Psychology and Theatre Studies.